Single-Molecule Patch-Clamp FRET Imaging Microscopy in Living Cells

描述（由申请人提供）：我们建议同时使用单分子光谱成像和单通道电流记录来询问单个离子通道构象运动的动力学，以更好地了解细胞膜通道功能的机制。为了满足医学生命科学的主要方法和技术需求和挑战，我们的提案的主要目标是解决自膜片钳电生理技术展示以来的过去四十年来理解离子通道蛋白生物学功能和动力学的关键问题。具体而言，我们建议进行系统的技术开发和演示。我们的项目包括三个主要目标：(1)实现对控制NMDA受体开合活性的亚基构象变化的实验理解；(2)解决NMDA受体在脱敏和失活过程中的构象变化；(3)展示单分子离子通道动力学研究的高时间分辨率。我们提出的技术方法，膜片钳共聚焦单分子荧光成像显微镜，将适用于各种细胞中的各种离子通道蛋白和受体，包括所有传统上通过传统膜片钳电流测量研究的细胞系统和可以通过荧光探测的蛋白质。我们将使用活细胞中的n -甲基- d -天冬氨酸（NMDA）受体作为模型系统来展示我们的创新物理技术及其在生命科学和医学研究中的前所未有的应用。NMDA受体介导的钙电流的动态对大脑的正常功能至关重要。NMDA受体活性的时间行为受激动剂结合和解离后蛋白质动态构象变化的调节。识别受体的构象运动是理解受体功能机制的关键。受体的不均匀构象运动控制通道的激活、失活、脱敏和失活，进而形成钙瞬态。

DESCRIPTION (provided by applicant): We propose to use single-molecule spectroscopy imaging simultaneously with single-channel current recording to interrogate the dynamics in the conformational motions of a single ion-channel, to better understand the mechanism of channel function at the cell membranes. To meet a major methodology and technical need and challenge in the medical life sciences, the primary goal of our proposal is to solve a critical problem that holds understanding ion channel protein biological function and dynamics over the last four decades ever since the patch-clamp electrophysiological technique demonstrated. Specifically, we propose to conduct a systematic technical development and demonstration. Our project consists of three primary aims: (1) Achieve an experimental understanding of the sub-unit conformational changes that control the open-close activity of the NMDA receptor; (2) Resolve the conformational changes of the NMDA receptor in desensitization and inactivation; and (3) Demonstrate high time resolution for single-molecule ion channel dynamics studies. Our proposed technical approach, patch-clamp confocal single-molecule fluorescence imaging microscopy, will be applicable for a broad range of ion channel proteins and receptors in various cells, including all of the cellular systems that have been traditionally studied by conventional patch-clamp electric current measurements and proteins that can be probed by fluorescence. We will use the N-methyl-D-aspartate (NMDA) receptor in living cells as a model system to demonstrate our innovative physical technique and its unprecedented applications in life sciences and medical researchers. The dynamics of NMDA receptor mediated calcium currents is crucial to the normal function of the brain. The temporal behavior of NMDA receptor activity is regulated by the dynamic conformational changes of the protein in response to agonist binding and dissociation. Identifying the conformational motions of the receptor is critical for understanding the mechanisms of receptor function. Inhomogeneous conformational motions of the receptor control the activation, inactivation, desensitization and deactivation of the channel that in turn shape the calcium transients.